ABSTRACT

Purpose

To develop a liquid formulation for IgMs to survive physical stress and storage.

Methods

Stabilizing formulations for 8 monoclonal immunoglobulin (IgMs) were found using differential scanning calorimetry (DSC). In these formulations, the IgMs were subjected to stress and storage and analyzed by size exclusion chromatography and fluorescence activated cell sorting. Structure was analyzed using small-angle X-ray scattering (SAXS).

Results

The highest conformational stability was found near the isoelectric point and further enhanced by addition of sorbitol, sucrose and glycine. For 2 IgMs, the pH optimum for conformational and storage stability did not correspond. Lowering the pH led to the desired storage stability. Optimized formulations prevented aggregation and fragmentation from shear stress, freeze-thaw cycles, accelerated storage and real time storage at 4°C and −20°C for 12 months. Optimized formulations also preserved immunoreactivity for 12 months. SAXS indicated that IgM in stabilizing conditions was closer to the structural IgM model (2RCJ) and less susceptible for aggregation.

Conclusions

A long-term stabilizing formulation for 8 IgMs was found comprising 20% sorbitol and 1 M glycine at pH 5.0–5.5 which may have broad utility for other IgMs. Formulation development using DSC and accelerated storage was evaluated in this study and may be used for other proteins.

KEY WORDS

conformational stability DSC formulation IgM protein aggregation

Electronic supplementary material

The online version of this article (doi:10.1007/s11095-012-0914-2) contains supplementary material, which is available to authorized users.

Notes

ACKNOWLEDGMENTS AND DISCLOSURES

The authors would like to gratefully thank Yih Yean Lee, Hui Ching Hia, Luo Weiwen, WenYan Lee and Hui Theng Gan for their help with producing and purifying the IgMs. Furthermore, the authors would like to thank Hoi Kong Meng for his help with SEC-SLS and Christopher Tan for his help with SEC-UV. We also thank Vanessa Ding for providing hESC for experiments. This work was supported by the Biomedical Research Council of A*STAR (Agency for Science, Technology and Research), Singapore. Rupert Tscheliessnig received a research grant from ACIB. ACIB is supported by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (BMVIT), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT – Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. We acknowledge the Department of Energy (DOE) Integrated Diffraction Analysis (IDAT) grant contract number DE-AC02-05CH11231.

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